Thiolactones

ABSTRACT

This invention provides new compounds, pharmaceutical compositions and diagnostic kits comprising such compounds, and methods of using such compounds for inhibiting NAALADase enzyme activity, detecting diseases where NAALADase levels are altered, inhibiting angiogenesis, effecting a TGF-β activity or a neuronal activity, and treating a glutamate abnormality, a compulsive disorder, neuropathy, pain, a prostate disease, cancer, Huntington&#39;s disease, diabetes, a retinal disorder or glaucoma.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/450,648, filed Mar. 3, 2003, the entire contents ofwhich are herein incorporated by reference.

This invention provides new compounds, pharmaceutical compositions anddiagnostic kits comprising such compounds, and methods of using suchcompounds for inhibiting NAALADase enzyme activity, detecting diseaseswhere NAALADase levels are altered, inhibiting angiogenesis, effecting aTGF-β activity or a neuronal activity, and treating a glutamateabnormality, a compulsive disorder, neuropathy, pain, a prostatedisease, cancer, Huntington's disease, diabetes, a retinal disorder orglaucoma.

The NAALADase enzyme, also known as prostate specific membrane antigen(“PSMA”) and human glutamate carboxypeptidase II (“GCP II”), catalyzesthe hydrolysis of the neuropeptide N-acetyl-aspartyl-glutamate (“NAAG”)to N-acetyl-aspartate (“NAA”) and glutamate. Based upon amino acidsequence homology, NAALADase has been assigned to the M28 family ofpeptidases.

Studies suggest that inhibitors of NAALADase may be useful in treatingischemia, spinal cord injury, demyelinating diseases, Parkinson'sdisease, Amyotrophic Lateral Sclerosis (“ALS”), Huntington's disease,alcohol dependence, nicotine dependence, cocaine dependence, opioiddependence, cancer, neuropathy, pain and schizophrenia, and ininhibiting angiogenesis. In view of their potential therapeuticapplications, a need exists for new NAALADase inhibitors and prodrugsthereof.

SUMMARY OF THE INVENTION

This invention provides a compound of formula I, II or III

or a pharmaceutically acceptable equivalent, an optical isomer or amixture of isomers of the compound, wherein:

X is C₁-C₄ alkylene, C₂-C₄ alkenylene, C₂-C₄ alkynylene, C₃-C₈cycloalkylene, C₅-C₇ cycloalkenylene or Ar, wherein the alkylene,alkenylene, alkynylene, cycloalkylene or cycloalkenylene isunsubstituted or substituted with one or more substituent(s);

L is a bond, —CR¹R²—, —O—, —S—, —SO₂— or —NR¹—;

Y is —O—, —S—, —CR³R⁴— or —NR³—;

Z is —(CR⁵R⁶)_(n)—;

n is 1, 2, 3 or 4;

Ar is a bivalent aryl or heteroaryl radical that is unsubstituted orsubstituted with one or more substituent(s);

R¹, R², R³, R⁴, R⁵ and R⁶ are independently hydrogen, C₁-C₄ alkyl orC₂-C₄ alkenyl, wherein the alkyl or alkenyl is unsubstituted orsubstituted with one or more substituent(s);

R⁷ is hydrogen, phenyl, phenylethyl or benzyl wherein the phenyl,phenylethyl or benzyl is unsubstituted or substituted with one or moresubstituent(s); and

R⁸, R⁹, R¹⁰ and R¹¹ are independently hydrogen, carboxy, hydroxy, halo,nitro, cyano, C₁-C₄ alkyl or C₁-C₄ alkoxy.

This invention further provides a pharmaceutical composition and adiagnostic kit comprising the compound, and a method of using thecompound for inhibiting NAALADase enzyme activity, detecting a diseasewhere NAALADase levels are altered, inhibiting angiogenesis, effecting aTGF-β activity or a neuronal activity, and treating a glutamateabnormality, a compulsive disorder, neuropathy, pain, a prostatedisease, cancer, Huntington's disease, diabetes, a retinal disorder orglaucoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a 27,000× magnified photograph of a retinal blood vessel froma control, non-diabetic rat.

FIG. 2 is a 27,000× magnified photograph of a retinal blood vessel froma diabetic rat after six months of treatment with a vehicle.

FIG. 3 is a 27,000× magnified photograph of a retinal blood vessel froma diabetic rat after six months of treatment with Compound B.

FIG. 4 is a graph plotting the time courses of tail-flick responses ofmice treated with a placebo, a NAALADase inhibitor, morphine, or aNAALADase inhibitor with morphine.

FIG. 5 is a bar graph plotting the mean±S.E.M. Area Under Curve (AUC)values.

DETAILED DESCRIPTION Definitions

“Compound B” refers to 2-(3-sulfanylpropyl)pentanedioic acid.

“Compound D” refers to 2-(2-sulfanylethyl)pentanedioic acid.

“Compound E” refers to3-carboxy-alpha-(3-mercaptopropyl)benzenepropanoic acid.

“Compound F” refers to3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)benzenepropanoicacid.

“Alkyl” refers to a univalent, saturated straight or branched chainhydrocarbon radical. Examples include, without limitation, methyl,ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl andn-hexyl.

“Alkylene” refers to a bivalent, saturated straight or branched chainhydrocarbon radical.

“Alkenyl” refers to a univalent, unsaturated straight or branched chainhydrocarbon radical comprising at least one carbon to carbon doublebond. Examples include, without limitation, ethenyl, propenyl,iso-propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyl andn-hexenyl.

“Alkenylene” refers to a bivalent, unsaturated straight or branchedchain hydrocarbon radical comprising at least one carbon to carbondouble bond.

“Alkynyl” refers to a univalent, unsaturated straight or branched chainhydrocarbon radical comprising at least one carbon to carbon triplebond. Examples include, without limitation, ethynyl, propynyl,iso-propynyl, butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl.

“Alkynylene” refers to a bivalent, unsaturated straight or branchedchain hydrocarbon radical comprising at least one carbon to carbontriple bond.

“Cycloalkyl” refers to a univalent, cyclic alkyl radical. Examplesinclude, without limitation, cyclobutyl, cycopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

“Cycloalkylene” refers to a bivalent, cyclic alkyl radical.

“Cycloalkenyl” refers to a univalent, cyclic alkenyl radical. Examplesinclude, without limitation, cyclopentenyl, cyclohexenyl, cycloheptenyland cyclooctenyl.

“Cycloalkenylene” refers to a bivalent, cyclic alkenyl radical.

“Alkoxy” refers to an alkyl group bonded through an oxygen linkage.

“Alkenoxy” refers to an alkenyl group bonded through an oxygen linkage.

“Aryl” refers to a cyclic aromatic hydrocarbon moiety having one or moreclosed ring(s). Examples include, without limitation, phenyl, benzyl,naphthyl, anthracenyl, phenanthracenyl and biphenyl.

“Heteroaryl” refers to a cyclic aromatic moiety having one or moreclosed rings with one or more heteroatom(s) (for example, sulfur,nitrogen or oxygen) in at least one ring. Examples include, withoutlimitation, pyrryl, furanyl, thienyl, pyridinyl, oxazolyl, thiazolyl,benzofuranyl, benzothienyl, benzofuranyl and benzothienyl.

“Carbocycle” refers to a hydrocarbon, cyclic moiety having one or moreclosed ring(s) that is/are alicyclic, aromatic, fused and/or bridged.Examples include, without limitation, cyclopropane, cyclobutane,cyclopentane, cyclohexane, cycloheptane, cyclopentene, cyclohexene,cycloheptene, cyclooctene, benzyl, naphthene, anthracene,phenanthracene, biphenyl and pyrene.

“Heterocycle” refers to a cyclic moiety having one or more closed ringsthat is/are alicyclic, aromatic, fused and/or bridged, with one or moreheteroatoms (for example, sulfur, nitrogen or oxygen) in at least one ofthe rings. Examples include, without limitation, pyrrolidine, pyrrole,thiazole, thiophene, piperidine, pyridine, isoxazolidine and isoxazole.

“Halo” refers to a fluoro, chloro, bromo or iodo radical.

“Isosteres” refer to elements, functional groups, substituents,molecules or ions having different molecular formulae but exhibitingsimilar or identical physical properties. For example, tetrazole is anisostere of carboxylic acid because it mimics the properties ofcarboxylic acid even though they have different molecular formulae.Typically, two isosteric molecules have similar or identical volumes andshapes. Ideally, isosteric molecules should be isomorphic and able toco-crystallize. Other physical properties that isosteric moleculesusually share include boiling point, density, viscosity and thermalconductivity. However, certain properties may be different: dipolarmoments, polarity, polarization, size and shape since the externalorbitals may be hybridized differently. The term “isosteres” encompasses“bioisosteres.”

“Bioisosteres” are isosteres that, in addition to their physicalsimilarities, share some common biological properties. Typically,bioisosteres interact with the same recognition site or produce broadlysimilar biological effects.

“Effective amount” refers to the amount required to produce a desiredeffect, for example, to inhibit NAALADase enzyme activity, to treat aglutamate abnormality, to effect a neuronal activity, to treat aprostate disease, to treat cancer, to inhibit angiogenesis, to effect aTGF-β activity, to treat Huntington's disease, to treat diabetes, totreat a retinal disorder or to treat glaucoma.

“Metabolite” refers to a substance produced by metabolism or by ametabolic process.

“NAAG” refers to N-acetyl-aspartyl-glutamate, an important peptidecomponent of the brain, with levels comparable to the major inhibitorneurotransmitter gamma-aminobutyric acid (GABA). NAAG isneuron-specific, present in synaptic vesicles and released upon neuronalstimulation in several systems presumed to be glutamatergic. Studiessuggest that NAAG may function as a neurotransmitter and/orneuromodulator in the central nervous system, or as a precursor of theneurotransmitter glutamate. In addition, NAAG is an agonist at group IImetabotropic glutamate receptors, specifically mGluR3 receptors; whenattached to a moiety capable of inhibiting NAALADase, it is expectedthat metabotropic glutamate receptor ligands will provide potent andspecific NAALADase inhibitors.

“NAALADase” refers to N-acetylated α-linked acidic dipeptidase, amembrane bound metallopeptidase which catabolizes NAAG toN-acetylaspartate (“NAA”) and glutamate (“GLU”):

NAALADase has been assigned to the M28 peptidase family and is alsocalled PSMA or human GCP II, EC number 3.4.17.21. It is believed thatNAALADase is a co-catalytic zinc/zinc metallopeptidase. NAALADase showsa high affinity for NAAG with a Km of 540 nM. If NAAG is a bioactivepeptide, then NAALADase may serve to inactivate NAAG'S synaptic action.Alternatively, if NAAG functions as a precursor for glutamate, theprimary function of NAALADase may be to regulate synaptic glutamateavailability.

“Inhibition,” in the context of enzymes, refers to reversible enzymeinhibition such as competitive, uncompetitive and non-competitiveinhibition. Competitive, uncompetitive and non-competitive inhibitioncan be distinguished by the effects of an inhibitor on the reactionkinetics of an enzyme. Competitive inhibition occurs when the inhibitorcombines reversibly with the enzyme in such a way that it competes witha normal substrate for binding at the active site. The affinity betweenthe inhibitor and the enzyme may be measured by the inhibitor constant,K_(i), which is defined as:$K_{i} = \frac{\lbrack E\rbrack\lbrack I\rbrack}{\lbrack{EI}\rbrack}$wherein [E] is the concentration of the enzyme, [I] is the concentrationof the inhibitor, and [EI] is the concentration of the enzyme-inhibitorcomplex formed by the reaction of the enzyme with the inhibitor. Unlessotherwise specified, K_(i) refers to the affinity between the inventivecompounds and NAALADase. Embodiments include a K_(i) of less than 100μM, less than 10 μM or less than 1 μM, as determined using anyappropriate assay known in the art. “IC₅₀” is a related term used todefine the concentration or amount of a compound that is required tocause a 50% inhibition of the target enzyme.

“Pharmaceutically acceptable carrier” refers to a pharmaceuticallyacceptable material, composition or vehicle, such as a liquid or solidfiller, diluent, excipient or solvent encapsulating material, involvedin carrying or transporting the subject compound from one organ, orportion of the body, to another organ or portion of the body. Eachcarrier is “acceptable” in the sense of being compatible with the otheringredients of the formulation and suitable for use with the patient.Examples of materials that can serve as a pharmaceutically acceptablecarrier include, without limitation: (1) sugars, such as lactose,glucose and sucrose; (2) starches, such as corn starch and potatostarch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such ascocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

“Pharmaceutically acceptable equivalent” includes, without limitation,pharmaceutically acceptable salts, hydrates, solvates, metabolites,prodrugs and isosteres. Many pharmaceutically acceptable equivalents areexpected to have the same or similar in vitro or in vivo activity as thecompounds of the invention.

“Pharmaceutically acceptable salt” refers to an acid or base salt of theinventive compounds, which salt possesses the desired pharmacologicalactivity and is neither biologically nor otherwise undesirable. The saltcan be formed with acids that include, without limitation, acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfatebutyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloridehydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,thiocyanate, tosylate and undecanoate. Examples of a base salt include,without limitation, ammonium salts, alkali metal salts such as sodiumand potassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such as arginineand lysine. In some embodiments, the basic nitrogen-containing groupscan be quarternized with agents including lower alkyl halides such asmethyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkylsulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides such as phenethyl bromides.

“Prodrug” refers to a derivative of the inventive compounds thatundergoes biotransformation, such as metabolism, before exhibiting itspharmacological effect(s). The prodrug is formulated with theobjective(s) of improved chemical stability, improved patient acceptanceand compliance, improved bioavailability, prolonged duration of action,improved organ selectivity, improved formulation (e.g., increasedhydrosolubility), and/or decreased side effects (e.g., toxicity). Theprodrug can be readily prepared from the inventive compounds usingconventional methods, such as that described in BURGER'S MEDICINALCHEMISTRY AND DRUG CHEMISTRY, Fifth Ed., Vol. 1, pp. 172-178, 949-982(1995).

“Derivative” refers to a substance produced from another substanceeither directly or by modification or partial substitution.

“Radiosensitizer” refers to a low molecular weight compound administeredto animals in therapeutically effective amounts to promote the treatmentof diseases that are treatable with electromagnetic radiation. Diseasesthat are treatable with electromagnetic radiation include, withoutlimitation, neoplastic diseases, benign and malignant tumors, andcancerous cells. Electromagnetic radiation treatment of other diseasesnot listed herein are also contemplated by this invention.

“Electromagnetic radiation” includes, without limitation, radiationhaving the wavelength of 10⁻²⁰ to 10⁰ meters. Examples include, withoutlimitation, gamma radiation (10⁻²⁰ to 10⁻¹³ m), X-ray radiation (10⁻¹¹to 10⁻⁹ m), ultraviolet light (10 nm to 400 nm), visible light (400 nmto 700 nm), infrared radiation (700 nm to 1.0 mm) and microwaveradiation (1 mm to 30 cm).

“Isomers” refer to compounds having the same number and kind of atoms,and hence the same molecular weight, but differing with respect to thearrangement or configuration of the atoms.

“Stereoisomers” refer to isomers that differ only in the arrangement ofthe atoms in space.

“Optical isomers” refer to enantiomers or diastereoisomers.

“Diastereoisomers” refer to stereoisomers that are not mirror images ofeach other. Diastereoisomers occur in compounds having two or moreasymmetric carbon atoms; thus, such compounds have 2^(n) opticalisomers, where n is the number of asymmetric carbon atoms.

“Enantiomers” refers to stereoisomers that are non-superimposable mirrorimages of one another.

“Enantiomer-enriched” refers to a mixture in which one enantiomerpredominates.

“Racemic mixture” refers to a mixture containing equal parts ofindividual enantiomers.

“Non-racemic mixture” refers to a mixture containing unequal parts ofindividual enantiomers.

“Angiogenesis” refers to the process whereby new capillaries are formed.“Inhibition” of angiogenesis may be measured by many parameters and, forexample, may be assessed by delayed appearance of neovascularstructures, slowed development of neovascular structures, decreasedoccurrence of neovascular structures, slowed or decreased severity ofangiogenesis-dependent disease effects, arrested angiogenic growth, orregression of previous angiogenic growth. In the extreme, completeinhibition is referred to herein as prevention. In relation toangiogenesis or angiogenic growth, “prevention” refers to no substantialangiogenesis or angiogenic growth if none has previously occurred, or nosubstantial additional angiogenesis or angiogenic growth if growth haspreviously occurred.

“Angiogenesis-dependent disease” includes, without limitation,rheumatoid arthritis, cardiovascular diseases, neovascular diseases ofthe eye, peripheral vascular disorders, dermatologic ulcers andcancerous tumor growth, invasion and metastasis.

“Animal” refers to a living organism having sensation and the power ofvoluntary movement, and which requires for its existence oxygen andorganic food. Examples include, without limitation, members of thehuman, equine, porcine, bovine, murine, canine and feline species. Inthe case of a human, an “animal” may also be referred to as a “patient.”

“Mammal” refers to a warm-blooded vertebrate animal.

“Anxiety” includes, without limitation, the emotional state consistingof psychophysiological responses to anticipation of unreal or imagineddanger, ostensibly resulting from unrecognized intrapsychic conflict.Physiological concomitants include increased heart rate, alteredrespiration rate, sweating, trembling, weakness, and fatigue;psychological concomitants include feelings of impending danger,powerlessness, apprehension, and tension. Dorland's Illustrated MedicalDictionary, W.B. Saunders Co., 27th ed. (1988).

“Anxiety Disorder” includes, without limitation, mental disorders inwhich anxiety and avoidance behavior predominate. Dorland's IllustratedMedical Dictionary, W.B. Saunders Co., 27th ed. (1988). Examplesinclude, without limitation, panic attack, agoraphobia, panic disorder,acute stress disorder, chronic stress disorder, specific phobia, simplephobia, social phobia, substance induced anxiety disorder, organicanxiety disorder, obsessive compulsive disorder, post-traumatic stressdisorder, generalized anxiety disorder, and anxiety disorder NOS. Otheranxiety disorders are characterized in Diagnostic and Statistical Manualof Mental Disorders (American Psychiatric Association 4th ed. 1994).

“Attention Deficit Disorder” (“ADD”) refers to a disorder characterizedby developmentally inappropriate inattention and impulsiveness, with orwithout hyperactivity. “Inattention” means a failure to finish tasksstarted, easily distracted, seeming lack of attention, and difficultyconcentrating on tasks requiring sustained attention. “Impulsiveness”means acting before thinking, difficulty taking turns, problemsorganizing work, and constant shifting from one activity to another.“Hyperactivity” means difficulty staying seated and sitting still, andrunning or climbing excessively.

“Cancer” includes, without limitation, ACTH-producing tumors, acutelymphocytic leukemia, acute nonlymphocytic leukemia, cancer of theadrenal cortex, bladder cancer, brain cancer, breast cancer, cervixcancer, chronic lymphocytic leukemia, chronic myelocytic leukemia,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cellleukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma,kidney cancer, liver cancer, lung cancer (small and/or non-small cell),malignant peritoneal effusion, malignant pleural effusion, melanoma,mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma,osteosarcoma, ovary cancer, ovary (germ cell) cancer, pancreatic cancer,penis cancer, prostate cancer, retinoblastoma, skin cancer, soft-tissuesarcoma, squamous cell carcinomas, stomach cancer, testicular cancer,thyroid cancer, trophoblastic neoplasms, cancer of the uterus, vaginalcancer, cancer of the vulva, and Wilm's tumor.

“Compulsive disorder” refers to any disorder characterized byirresistible impulsive behavior. Examples of compulsive disordersinclude, without limitation, substance dependence, eating disorder,pathological gambling, ADD and Tourette's syndrome.

“Demyelinating disease” refers to any disease involving damage to orremoval of the myelin sheath naturally surrounding nerve tissue, such asthat defined in U.S. Pat. No. 5,859,046 and International PublicationNo. WO 98/03178, herein incorporated by reference. Examples include,without limitation, peripheral demyelinating diseases (such asGuillain-Barré syndrome, peripheral neuropathies and Charcot-Marie Toothdisease) and central demyelinating diseases (such as multiplesclerosis).

“Disease” refers to any deviation from or interruption of the normalstructure or function of any part, organ or system (or combinations) ofthe body that is manifested by a characteristic set of symptoms andsigns and whose etiology, pathology, and prognosis may be known orunknown. Dorland's Illustrated Medical Dictionary, (W.B. Saunders Co.27th ed. 1988).

“Disorder” refers to any derangement or abnormality of function; amorbid physical or mental state. Dorland's Illustrated MedicalDictionary, (W.B. Saunders Co. 27th ed. 1988).

“Eating disorder” refers to compulsive overeating, obesity or severeobesity. Obesity means body weight of 20% over standard height-weighttables. Severe obesity means over 100% overweight.

“Glaucoma” includes, without limitation, chronic (idiopathic) open-angleglaucoma (e.g., high-pressure, normal-pressure); pupillary blockglaucoma (e.g., acute angle-closure, subacute angle-closure, chronicangle-closure, combined-mechanism); developmental glaucoma (e.g.,congenital (infantile), juvenile, Anxenfeld-Rieger syndrome, Peters'anomaly, Aniridia); glaucoma associated with other ocular disorders(e.g., glaucoma associated with disorder of the corneal endothelium,iris, ciliary body, lens, retina, choroid or vitreous); glaucomaassociated with elevated episcleral venous pressure (e.g., systemicdiseases with associated elevated intraocular pressure and glaucoma,corticosteroid-induced glaucoma); glaucoma associated with inflammationand trauma (e.g., glaucoma associated with keratitis, episcleritis,scleritis, uveitis, ocular trauma and hemorrhage); glaucoma followingintraocular surgery (e.g., ciliary block (malignant) glaucoma, glaucomain aphakia and pseudophakia, glaucoma associated with corneal surgery,glaucoma associated with vitreoretinal surgery).

“Glutamate abnormality” refers to any disease, disorder, or condition inwhich glutamate is implicated, including a pathological conditioninvolving elevated levels of glutamate. Examples of a glutamateabnormality include, without limitation, compulsive disorder, spinalcord injury, epilepsy, stroke, ischemia, demyelinating disease,Alzheimer's disease, Parkinson's disease, ALS, Huntington's disease,schizophrenia, pain, peripheral neuropathy (including but not limited todiabetic neuropathy), traumatic brain injury, neuronal insult,inflammatory disease, anxiety, anxiety disorder, memory impairment,glaucoma and retinal disorder.

“Ischemia” refers to localized tissue anemia due to obstruction of theinflow of arterial blood. Global ischemia occurs when blood flow ceasesfor a period of time, as may result from cardiac arrest. Focal ischemiaoccurs when a portion of the body, such as the brain, is deprived of itsnormal blood supply, such as may result from thromboembolytic occlusionof a cerebral vessel, traumatic head injury, edema or brain tumor. Evenif transient, both global and focal ischemia can produce widespreadneuronal damage. Although nerve tissue damage occurs over hours or evendays following the onset of ischemia, some permanent nerve tissue damagemay develop in the initial minutes following cessation of blood flow tothe brain. Much of this damage is attributed to glutamate toxicity andsecondary consequences of reperfusion of the tissue, such as the releaseof vasoactive products by damaged endothelium, and the release ofcytotoxic products, such as free radicals and leukotrienes, by thedamaged tissue.

“Memory impairment” refers to a diminished mental registration,retention or recall of past experiences, knowledge, ideas, sensations,thoughts or impressions. Memory impairment may affect short andlong-term information retention, facility with spatial relationships,memory (rehearsal) strategies, and verbal retrieval and production.Common causes of memory impairment are age, severe head trauma, brainanoxia or ischemia, alcoholic-nutritional diseases, drug intoxicationsand neurodegenerative diseases. For example, memory impairment is acommon feature of neurodegenerative diseases such as Alzheimer's diseaseand senile dementia of the Alzheimer type. Memory impairment also occurswith other kinds of dementia, such as multi-infarct dementia, a seniledementia caused by cerebrovascular deficiency, and the Lewy-body variantof Alzheimer's disease with or without association with Parkinson'sdisease. Creutzfeldt-Jakob disease is a rare dementia with which memoryimpairment is associated. It is a spongiform encephalopathy caused bythe prion protein; it may be transmitted from other sufferers or mayarise from gene mutations. Loss of memory is also a common feature ofbrain-damaged patients. Brain damage may occur, for example, after aclassical stroke or as a result of an anaesthetic accident, head trauma,hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin(B₁, thiamine and B₁₂) deficiency, or excessive alcohol use. Korsakoff samnesic psychosis is a rare disorder characterized by profound memoryloss and confabulation, whereby the patient invents stories to concealhis or her memory loss. It is frequently associated with excessivealcohol intake. Memory impairment may furthermore be age-associated; theability to recall information such as names, places and words seems todecrease with increasing age. Transient memory loss may also occur inpatients, suffering from a major depressive disorder, afterelectro-convulsive therapy.

“Mental disorder” refers to any clinically significant behavioral orpsychological syndrome characterized by the presence of distressingsymptoms or significant impairment of functioning. Mental disorders areassumed to result from some psychological or organic dysfunction of theindividual; the concept does not include disturbances that areessentially conflicts between the individual and society (socialdeviance).

“Metastasis” refers to “[t]he ability of cells of a cancer todisseminate and form new foci of growth at noncontiguous sites (i.e., toform metastases).” See Hill, R. P, “Metastasis”, The Basic Science ofOncology, Tannock et al., Eds., McGraw-Hill, New York, pp. 178-195(1992), herein incorporated by reference. “The transition from in situtumor growth to metastatic disease is defined by the ability of tumorcells of the primary site to invade local tissues and to cross tissuebarriers . . . . To initiate the metastatic process, carcinoma cellsmust first penetrate the epithelial basement membrane and then invadethe interstitial stroma. For distant metastases, intravasation requirestumor cell invasion of the subendothelial basement membrane that mustalso be negotiated during tumor cell extravasation . . . . Thedevelopment of malignancy is also associated with tumor-inducedangiogenesis [which] not only allows for expansion of the primarytumors, but also permits easy access to the vascular compartment due todefects in the basement membranes of newly formed vessels.” SeeAznavoorian et al., Cancer (1993) 71:1368-1383, herein incorporated byreference.

“Neuropathy” refers to any disease or malfunction of the nerves.Neuropathy includes, without limitation, peripheral neuropathy, diabeticneuropathy, autonomic neuropathy and mononeuropathy. Peripheralneuropathy may be idiopathic or induced by any causes including diseases(for example, amyloidosis, alcoholism, HIV, syphilis, virus, autoimmunedisorder, cancer, porphyria, arachnoiditis, post herpetic neuralgia,Guillain-Barré syndrome, diabetes including type I and type IIdiabetes), chemicals (for example, toxins, lead, dapsone, vitamins,paclitaxel chemotherapy, HAART therapy) and physical injuries to aparticular nerve or nerve plexus (for example, trauma, compression,constriction).

“Neuroprotective” refers to the effect of reducing, arresting orameliorating nervous insult, and protecting, resuscitating or revivingnervous tissue that has suffered nervous insult.

“Nervous insult” refers to any damage to nervous tissue and anydisability or death resulting therefrom. The cause of nervous insult maybe metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, andincludes, without limitation, ischemia, hypoxia, cerebrovascularaccident, trauma, surgery, pressure, mass effect, hemorrhage, radiation,vasospasm, neurodegenerative disease, neurodegenerative process,infection, Parkinson's disease, ALS, myelination/demyelinationprocesses, epilepsy, cognitive disorder, glutamate abnormality andsecondary effects thereof.

“Nervous tissue” refers to the various components that make up thenervous system, including without limitation neurons, neural supportcells, glia, Schwann cells, vasculature contained within and supplyingthese structures, the central nervous system, the brain, the brain stem,the spinal cord, the junction of the central nervous system with theperipheral nervous system, the peripheral nervous system and alliedstructures.

“Pain” refers to localized sensations of discomfort, distress or agony,resulting from the stimulation of specialized nerve endings. It servesas a protective mechanism insofar as it induces the sufferer to removeor withdraw from the source. Dorland's Illustrated Medical Dictionary,(W.B. Saunders Co. 27th ed. 1988). Examples of pain include, withoutlimitation, acute, chronic, cancer, burn, incisional, inflammatory,neuropathic and back pain.

“Neuropathic pain” refers to a condition of pain associated with a nerveinjury. Depending on the particular syndrome, the pain may be due toalterations of the brain or spinal cord or may be due to abnormalitiesin the nerve itself. Neuropathic pain may be idiopathic or induced byany causes including diseases (for example, amyloidosis, alcoholism,HIV, syphilis, virus, autoimmune disorder, cancer, porphyria,arachnoiditis, post herpetic neuralgia, Guillain-Barré syndrome,diabetes including type I and type II diabetes), chemicals (for example,toxins, lead, dapsone, vitamins, paclitaxel chemotherapy, HAART therapy)and physical injuries to a particular nerve or nerve plexus (forexample, trauma, compression, constriction).

“Pathological gambling” refers to a condition characterized by apreoccupation with gambling. Similar to psychoactive substance abuse,its effects include development of tolerance with a need to gambleprogressively larger amounts of money, withdrawal symptoms, andcontinued gambling despite severe negative effects on family andoccupation.

“Prostate disease” refers to any disease affecting the prostate.Examples of prostate disease include, without limitation, prostatecancer (e.g., adenocarcinoma and metastatic cancers of the prostate) andconditions characterized by abnormal growth of prostatic epithelialcells (e.g., benign prostatic hyperplasia).

“Retinal disorder” refers to vascular retinopathy, for example,hypertensive retinopathy, diabetic retinopathy (nonproliferative orproliferative), central retinal artery occlusion, or central retinalvein occlusion; age-related macular degeneration; retinal detachment; orretinitis pigmentosa.

“Schizophrenia” refers to a mental disorder or group of mental disorderscharacterized by disturbances in form and content of thought (looseningof associations, delusions, hallucinations), mood (blunted, flattened,inappropriate affect), sense of self and relationship to the externalworld (loss of ego boundaries, dereistic thinking, and autisticwithdrawal), and behavior (bizarre, apparently purposeless, andstereotyped activity or inactivity). Examples of schizophrenia include,without limitation, acute, ambulatory, borderline, catatonic, childhood,disorganized, hebephrenic, latent, nuclear, paranoid, paraphrenic,prepsychotic, process, pseudoneurotic, pseudopsychopathic, reactive,residual, schizo-affective and undifferentiated schizophrenia. Dorland'sIllustrated Medical Dictionary (W.B. Saunders Co. 27th ed. 1988).

“TGF-β” refers to transforming growth factor beta. TGF-β is recognizedas a prototype of multifunctional growth factors. It regulates variouscell and tissue functions, including cell growth and differentiation,angiogenesis, wound healing, immune function, extracellular matrixproduction, cell chemotaxis, apoptosis and hematopoiesis.

“TGF-β abnormality” refers to any disease, disorder or condition inwhich TGF-β is implicated, including diseases disorders and conditionscharacterized by an abnormal level of TGF-β.

“Abnormal level of TGF-β” refers to a measurable variance from normallevels of TGF-β, as determined by one of ordinary skill in the art usingknown techniques.

“Therapeutic window of opportunity” or “window” refers, in relation tostroke, to the maximal delay between the onset of stroke and theinitiation of efficacious therapy.

“Tourette's syndrome” refers to an autosomal multiple tic disordercharacterized by compulsive swearing, multiple muscle tics and loudnoises. Tics are brief, rapid, involuntary movements that can be simpleor complex; they are stereotyped and repetitive, but not rhythmic.Simple tics, such as eye blinking, often begin as nervous mannerisms.Complex tics often resemble fragments of normal behavior.

Unless otherwise defined in conjunction with a specific disease,disorder or condition, “treating” refers to:

(i) preventing a disease, disorder or condition from occurring in ananimal that may be predisposed to the disease, disorder and/or conditionbut has not yet been diagnosed as having it;

(ii) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and/or

(iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

“Treating ALS” refers to:

(i) preventing ALS from occurring in an animal that may be predisposedto ALS but has not yet been diagnosed as having it;

(ii) inhibiting ALS, e.g. arresting its development;

(iii) relieving ALS, e.g. causing regression of the disease, disorderand/or condition;

(iv) delaying onset of ALS or ALS symptom(s);

(v) slowing progression of ALS or ALS symptom(s);

(vi) prolonging survival of an animal suffering from ALS; and/or

(vii) attenuating ALS symptom(s).

“Treating Huntington's disease” refers to:

(i) preventing Huntington's disease from occurring in an animal that maybe predisposed to Huntington's disease but has not yet been diagnosed ashaving it;

(ii) inhibiting or slowing Huntington's disease, e.g. arresting itsdevelopment;

(iii) relieving Huntington's disease, e.g. causing its regression;

(iv) improving motor coordination in an animal having Huntington'sdisease; and/or

(v) prolonging survival of an animal having Huntington's disease.

“Treating substance dependence” refers to preventing relapse; reducingcraving; suppressing tolerance; preventing, inhibiting and/or relievingwithdrawal; attenuating sensitization; preventing, inhibiting (i.e.arresting development of) and/or relieving (i.e. causing regression of)substance-induced neurotoxicity; and/or preventing, inhibiting and/orrelieving fetal alcohol syndrome.

“Craving” refers to a strong desire for a substance and/or a compellingurge and/or an irresistible impulse to use a substance.

“Dependence” refers to a maladaptive pattern of substance use, leadingto clinically significant impairment or distress. Dependence istypically characterized by tolerance and/or withdrawal. Substances forwhich dependence may be developed include, without limitation,depressants (opioids, synthetic narcotics, barbiturates, glutethimide,methyprylon, ethchlorvynol, methaqualone, alcohol); anxiolytics(diazepam, chlordiazepoxide, alprazolam, oxazepam, temazepam);stimulants (amphetamine, methamphetamine, cocaine, nicotine); andhallucinogens (LSD, mescaline, peyote, marijuana).

“Opioid” refers to a narcotic analgesic that is either semi or fullysynthetic, including, but not limited to Codeine, Morphine, Heroin,Hydromorphone (Dilaudid), Oxycodone (Percodan), Oxymorphone (Numorphan),Hydrocodone (Vicodin), Meperidine (Demerol), Fentanyl, Methadone(Dolophine), Darvon, Talwin.

“Relapse” refers to a return to substance use after a period ofabstinence, often accompanied by reinstatement.

“Reinstatement” refers to a return to a preexisting level of use anddependence in a person who has resumed substance use following a periodof abstinence.

“Sensitization” refers to a condition in which the response to asubstance increases with repeated use.

“Tolerance” refers to an acquired reaction to a substance characterizedby diminished effect with continued use of the same dose and/or a needfor increased doses to achieve intoxication or desired effect previouslyachieved by lower doses. Both physiological and psychosocial factors maycontribute to the development of tolerance. With respect tophysiological tolerance, metabolic and/or functional tolerance maydevelop. By increasing the rate of metabolism of the substance, the bodymay be able to eliminate the substance more readily. Functionaltolerance is defined as a decrease in sensitivity of the central nervoussystem to the substance. “Opioid tolerance” includes without limitationthe failure of a steady dose of the drug to sustain the desiredpharmacological effect over time, i.e., the need to increase the drugdosage to maintain the original pharmacological effect.

“Withdrawal” refers to a syndrome characterized by untoward physicalchanges that occur following cessation of or reduction in substance use,or administration of a pharmacologic antagonist.

One of ordinary skill in the art will recognize that there arealternative nomenclatures, nosologies and classification systems for thediseases, disorders and conditions defined above, and that such systemsevolve with medical scientific progress.

Unless the context clearly dictates otherwise, the definitions ofsingular terms may be extrapolated to apply to their plural counterpartsas they appear in the application; likewise, the definitions of pluralterms may be extrapolated to apply to their singular counterparts asthey appear in the application.

Compounds

This invention provides a compound of formula I, II or III

or a pharmaceutically acceptable equivalent, an optical isomer or amixture of isomers of the compound, wherein:

X is C₁-C₄ alkylene, C₂-C₄ alkenylene, C₂-C₄ alkynylene, C₃-C₈cycloalkylene, C₅-C₇ cycloalkenylene or Ar, wherein the alkylene,alkenylene, alkynylene, cycloalkylene or cycloalkenylene isunsubstituted or substituted with one or more substituent(s);

L is a bond, —CR¹R²—, —O—, —S—, —SO₂— or —NR¹—;

Y is —O—, —S—, —CR³R⁴— or —NR³—;

Z is —(CR⁵R⁶)_(n)—;

n is 1, 2, 3 or 4;

Ar is a bivalent aryl or heteroaryl radical that is unsubstituted orsubstituted with one or more substituent(s);

R¹, R², R³, R⁴, R⁵ and R⁶ are independently hydrogen, C₁-C₄ alkyl orC₂-C₄ alkenyl, wherein the alkyl or alkenyl is unsubstituted orsubstituted with one or more substituent(s);

R⁷ is hydrogen, phenyl, phenylethyl or benzyl wherein the phenyl,phenylethyl or benzyl is unsubstituted or substituted with one or moresubstituent(s); and

R⁸, R⁹, R¹⁰ and R¹¹ are independently hydrogen, carboxy, hydroxy, halo,nitro, cyano, C₁-C₄ alkyl or C₁-C₄ alkoxy.

In one embodiment of the compound of formula I, when L is a bond and Xis ethyl, then Y is not —CR³R⁴—.

In another embodiment of the compound of formula I:

-   -   Y is —CR³R⁴—; and    -   n is 1 or 2.

In a further embodiment of the compound of formula I:

-   -   L is —CR¹R²—, —O—, —S— or NH;    -   X is C₁-C₂ alkylene or Ar; and    -   Ar is phenylene, biphenylene, benzylene or naphthylene, wherein        the phenylene, biphenylene, benzylene or naphthylene is        unsubstituted or substituted with one or more substituent(s)        independently selected from carboxy, halo, nitro, C₁-C₄ alkyl, ,        C₁-C₄ alkoxy, phenyl, phenoxy and benzyloxy.

In one embodiment of the compound of formula II:

-   -   L is a bond, —CR¹R²— or —O—; and    -   n is 2.

In another embodiment of the compound of formula II:

-   -   X is C₁-C₄ alkylene or Ar; and    -   Ar is phenylene, biphenylene or benzylene that is unsubstituted        or substituted with one or more substituent(s) independently        selected from carboxy, halo, nitro, C₁-C₄ alkyl, C₁-C₄ alkoxy,        phenoxy and benzyloxy.

In one embodiment of the compound of formula III:

-   -   R⁸, R⁹, R¹⁰ and R¹¹ are independently hydrogen or carboxy.

In another embodiment of the compound of formula III:

-   -   R⁷ is phenyl or benzyl substituted with one or more        substituent(s) independently selected from carboxy, halo, C₁-C₄        alkyl and C₁-C₄ alkoxy.

Examples of the one or more substituent(s) by which X, Ar, R¹, R², R³,R⁴, R⁵, R⁶ and R⁷ may be substituted include, without limitation: C₁-C₄alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, C₁-C₄ alkoxy, C₂-C₄ alkenyloxy,phenoxy, benzyloxy, hydroxy, carboxy, hydroperoxy, carbamido, carbamoyl,carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl,guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido,hydrazino, triazano, nitrilo, nitro, nitroso, isonitroso, nitrosamino,imino, nitrosimino, oxo, C₁-C₄ alkylthio, sulfamino, sulfamoyl, sulfeno,sulfhydryl, sulfinyl, sulfo, sulfonyl, thiocarboxy, thiocyano,isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl,perchloryl, trifluoromethyl, iodosyl, iodyl, phosphino, phosphinyl,phospho, phosphono, arsino, selanyl, disilanyl, siloxy, silyl, silyleneand carbocyclic and heterocyclic moieties.

The definition of any variable substituent at a particular location in amolecule is independent of its definitions elsewhere in that molecule.Substituents and substitution patterns on the inventive compounds can beselected by one of ordinary skill in the art to provide compounds thatare chemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

Since the inventive compounds may possess one or more asymmetric carboncenter(s), they may be capable of existing in the form of opticalisomers as well as in the form of racemic or non-racemic mixtures ofoptical isomers. The optical isomers can be obtained by resolution ofthe racemic mixtures according to conventional processes. One suchprocess entails formation of diastereoisomeric salts by treatment withan optically active acid or base, then separation of the mixture ofdiastereoisomers by crystallization, followed by liberation of theoptically active bases from the salts. Examples of appropriate acids aretartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric andcamphorsulfonic acid.

A different process for separating optical isomers involves the use of achiral chromatography column optimally chosen to maximize the separationof the enantiomers. Still another available process involves synthesisof covalent diastereoisomeric molecules, for example, esters, amides,acetals and ketals, by reacting the inventive compounds with anoptically active acid in an activated form, an optically active diol oran optically active isocyanate. The synthesized diastereoisomers can beseparated by conventional means such as chromatography, distillation,crystallization or sublimation, and then hydrolyzed to deliver theenantiomerically pure compound. In some cases hydrolysis to the “parent”optically active drug is not necessary prior to dosing the patient,since the compound can behave as a prodrug. The optically activecompounds of this invention likewise can be obtained by utilizingoptically active starting materials.

The compounds of this invention encompass individual optical isomers aswell as racemic and non-racemic mixtures. In some non-racemic mixtures,the R configuration may be enriched while in other non-racemic mixtures,the S configuration may be enriched enriched.

Examples of the inventive compounds include, without limitation, thecompounds set forth in TABLE I. TABLE I REPRESENTATIVE COMPOUNDSCompound Number Structure Name 1

3-(2-Oxo-tetrahydro- thiopyran-3-yl)- propionic acid 2

3-[(2-oxotetrahydro- 2H-thiopyran-3-yl) methyl]benzoic acid 3

3-(1-Oxo-isothio- chroman-8-yl)- benzoic acid 4

3-(1-Oxo-isothio- chroman-8-yloxy- methyl)-benzoic acid 5

3-(1-Oxo-3,4-dihydro- 1H-2-thia-9-aza- fluoren-9-yl)-benzoic acid

Methods of Use

While the inventive compounds are generally prodrugs of NAALADaseinhibitors, the inventive compounds may also exhibit NAALADaseinhibiting activity on their own. Whether by inhibiting NAALADaseactivity, by converting in vivo into compounds that inhibit NAALADaseactivity, or by another mechanism of action, the inventive compounds maybe useful in the following therapeutic applications.

Method for Inhibiting NAALADase Enzyme Activity

This invention further provides a method for inhibiting NAALADase enzymeactivity in an animal or a mammal, comprising administering to theanimal or mammal an effective amount of a compound of formula I, II orIII, as defined above.

Method for Treating Glutamate Abnormalities

Without being bound to any particular mechanism of action, the inventivecompounds may block glutamate release pre-synaptically withoutinteracting with post-synaptic glutamate receptors. Such compounds wouldbe devoid of the behavioral toxicities associated with post-synapticglutamate antagonists. Thus, this invention further provides a methodfor treating a glutamate abnormality in an animal or a mammal,comprising administering to the animal or mammal an effective amount ofa compound of formula I, II or III, as defined above.

The glutamate abnormality that is treated by the inventive method may beselected from compulsive disorder, stroke, ischemia, demyelinatingdisease, Parkinson's disease, ALS, Huntington's disease, schizophrenia,diabetic neuropathy, pain, anxiety, anxiety disorder, memory impairmentand glaucoma. In one embodiment, the inventive method is for treating acompulsive disorder selected from alcohol, nicotine, cocaine and opioiddependence. In another embodiment, the inventive method is for treatingopioid tolerance.

Stroke patients often experience a significant temporal delay betweenthe onset of ischemia and the initiation of therapy. Thus, there is aneed for neuroprotectants with a long therapeutic window of opportunity.It is expected that the inventive compounds have a therapeutic window ofopportunity of at least 1 hour. Accordingly, when the glutamateabnormality is stroke, the compound of the invention may be administeredto the animal or mammal for up to 60 minutes, 120 minutes or morefollowing onset of stroke.

Method for Effecting Neuronal Activities

This invention further provides a method for effecting a neuronalactivity in an animal or a mammal, comprising administering to theanimal or mammal an effective amount of a compound of formula I, II orIII, as defined above.

The neuronal activity that is effected by the inventive method may bestimulation of damaged neurons, promotion of neuronal regeneration,prevention of neurodegeneration or treatment of a neurological disorder.

Examples of a neurological disorder that is treated by the inventivemethod include, without limitation: trigeminal neuralgia;glossopharyngeal neuralgia; Bell's Palsy; myasthenia gravis; musculardystrophy; ALS; progressive muscular atrophy; progressive bulbarinherited muscular atrophy; herniated, ruptured or prolapsedinvertebrate disk syndromes; cervical spondylosis; plexus disorders;thoracic outlet destruction syndromes; neuropathy; pain; Alzheimer'sdisease; Parkinson's disease; ALS; and Huntington's disease.

In one embodiment, the inventive method is for treating a neurologicaldisorder selected from neuropathy (for example, peripheral neuropathy ordiabetic neuropathy), pain (for example, neuropathic pain such asneuropathic pain induced by diabetes), traumatic brain injury, physicaldamage to spinal cord, stroke associated with brain damage,demyelinating disease and neurological disorder relating toneurodegeneration.

When the neurological disorder is pain, the compound of the inventionmay be administered in combination with an effective amount of morphine.

Examples of a neurological disorder relating to neurodegenerationinclude, without limitation, Alzheimer's disease, Parkinson's diseaseand ALS.

Method for Treating Prostate Diseases

This invention further provides a method for treating a prostate diseasein an animal or a mammal, comprising administering to the animal ormammal an effective amount of a compound of formula I, II or III, asdefined above. In one embodiment, the prostate disease is prostatecancer.

Method for Treating Cancers

This invention further provides a method for treating cancer in ananimal or a mammal, comprising administering to the animal or mammal aneffective amount of a compound of formula I, II or III, as definedabove. In one embodiment, the cancer is in tissues where NAALADaseresides, such as the brain, kidney and testis.

Method for Inhibiting Angiogenesis

This invention further provides a method for inhibiting angiogenesis inan animal or a mammal, comprising administering to the animal or mammalan effective amount of a compound of formula I, II or III, as definedabove.

Angiogenesis may be necessary for fertility or metastasis of cancertumors, or may be related to an angiogenic-dependent disease. Thus, thisinvention further provides a method for treating an angiogenic-dependentdisease. Examples of an angiogenic-dependent disease include, withoutlimitation, rheumatoid arthritis, cardiovascular diseases, neovasculardiseases of the eye, peripheral vascular disorders, dermatologic ulcersand cancerous tumor growth, invasion or metastasis.

Method for Effecting TGF-β Activity

This invention further provides a method for effecting a TGF-β activityin an animal or a mammal, comprising administering to the animal ormammal an effective amount of a compound of formula I, II or III, asdefined above.

The effecting a TGF-β activity includes increasing, reducing orregulating TGF-β levels, and treating a TGF-β abnormality. Examples of aTGF-β abnormality that is treated by the inventive method includeneurodegenerative disorders, extra-cellular matrix formation disorders,cell-growth related diseases, infectious diseases, immune relateddiseases, epithelial tissue scarring, collagen vascular diseases,fibroproliferative disorders, connective tissue disorders, inflammation,inflammatory diseases, respiratory distress syndrome, infertility anddiabetes.

Examples of a neurodegenerative disorder include, without limitation,neural tissue damage resulting from ischemia, reperfusion injury,myelination or neurodegeneration.

Examples of a cell-growth related disorder include, without limitation,disorders affecting kidney cells, hematopoietic cells, lymphocytes,epithelial cells or endothelial cells.

Examples of an infectious disease include, without limitation, diseasescaused by a macrophage pathogen, particularly a macrophage pathogenselected from bacteria, yeast, fungi, viruses, protozoa, Trypanosomacruzi, Histoplasma capsulatum, Candida albicans, Candida parapsilosis,Cryptococcus neoformans, Salmonella, Pneumocystis, Toxoplasma, Listeria,Mycobacteria, Rickettsia and Leishmania. Examples of Mycobacteriainclude, without limitation, Mycobacterium tuberculosis andMycobacterium leprae. Examples of Toxoplasma include, withoutlimitation, Toxoplasma gondii. Examples of Rickettsia include, withoutlimitation, R. prowazekii, R. coronii and R. tsutsugamushi. Otherexamples of an infectious disease include single or multiple cutaneouslesions, mucosal disease, Chagas' disease, acquired immunodeficiencysyndrome (AIDS), toxoplasmosis, leishmaniasis, trypanosomiasis,shistosomiasis, cryptosporidiosis, Mycobacterium avium infections,Pneumocystis carinii pneumonia and leprosy.

Examples of an immune related disease include, without limitation,autoimmune disorders; impaired immune function; and immunosuppressionassociated with an infectious disease, particularly, trypanosomalinfection, viral infection, human immunosuppression virus, human T celllymphotropic virus (HTLV-1), lymphocytic choriomeningitis virus orhepatitis.

Examples of a collagen vascular disease include, without limitation,progressive systemic sclerosis (“PSS”), polymyositis, scleroderma,dermatomyositis, eosinophilic fascitis, morphea, Raynaud's syndrome,interstitial pulmonary fibrosis, scleroderma and systemic lupuserythematosus.

Examples of a fibroproliferative disorder include, without limitation,diabetic nephropathy, kidney disease, proliferative vitreoretinopathy,liver cirrhosis, biliary fibrosis and myelofibrosis. Examples of akidney disease include, without limitation, mesangial proliferativeglomerulonephritis, crescentic glomerulonephritis, diabetic neuropathy,renal interstitial fibrosis, renal fibrosis in transplant patientsreceiving cyclosporin, and HIV-associated nephropathy.

Examples of a connective tissue disorder include, without limitation,scleroderma, myelofibrosis, and hepatic, intraocular and pulmonaryfibrosis.

Without being limited to any particular mechanism of action, theinventive compounds may treat inflammatory diseases by regulating TGF-βand/or inhibiting myeloperoxidase. Examples of an inflammatory diseasethat is treated by the inventive method include, without limitation, adisease associated with PSS, polymyositis, scleroderma, dermatomyositis,eosinophilic fascitis, morphea, Raynaud's syndrome, interstitialpulmonary fibrosis, scleroderma, systemic lupus erythematosus, diabeticnephropathy, kidney disease, proliferative vitreoretinopathy, livercirrhosis, biliary fibrosis, myelofibrosis, mesangial proliferativeglomerulonephritis, crescentic glomerulonephritis, diabetic neuropathy,renal interstitial fibrosis, renal fibrosis in transplant patientsreceiving cyclosporin, and HIV-associated nephropathy.

Other uses associated with the inventive compounds' TGF-β regulatingproperties include:

stimulating growth of tissues, glands or organs, particularly growththat would enhance milk production or weight gain;

stimulating cell proliferation, particularly proliferation offibroblasts, mesenchymal cells or epithelial cells;

inhibiting cell growth, particularly of epithelial cells, endothelialcells, T and B lymphocytes or thymocytes;

inhibiting expression of adipose, skeletal muscle or hematopoieticphenotypes, neoplasms, non-cytocidal viral or other pathogenicinfections or autoimmune disorders;

mediating disease resistance or susceptibility;

suppressing cellular immune response;

inhibiting scar tissue formation, such as in skin or other epithelialtissue that has been damaged by wounds resulting from accidental injury,surgical operations, trauma-induced lacerations or other trauma, or bywounds involving the peritoneum for which the excessive connectivetissue formation is abdominal adhesions;

increasing the effectiveness of a vaccine, particularly a vaccine for anallergy towards, for example, dust or hayfever; and

inhibiting polyp formation.

Administration and Dosage

The inventive compounds may be administered by any means known to one ofordinary skill in the art. For example, the inventive compounds may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally, or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intraperitoneal, intrathecal, intraventricular,intrasternal, intracranial, and intraosseous injection and infusiontechniques. The exact administration protocol will vary depending uponvarious factors including the age, body weight, general health, sex anddiet of the patient; the determination of specific administrationprocedures would be routine to an one of ordinary skill in the art.

The inventive compounds may be administered by a single dose, multiplediscrete doses or continuous infusion. Pump means, particularlysubcutaneous pump means, are useful for continuous infusion.

Dose levels on the order of about 0.001 mg/kg/d to about 10,000 mg/kg/dof an inventive compound are useful for the inventive methods. In oneembodiment, the dose level is about 0.1 mg/kg/d to about 1,000 mg/kg/d.In another embodiment, the dose level is about 1 mg/kg/d to about 100mg/kg/d. The specific dose level for any particular patient will varydepending upon various factors, including the activity and the possibletoxicity of the specific compound employed; the age, body weight,general health, sex and diet of the patient; the time of administration;the rate of excretion; the drug combination; the severity of thecongestive heart failure; and the form of administration. Typically, invitro dosage-effect results provide useful guidance on the proper dosesfor patient administration. Studies in animal models are also helpful.The considerations for determining the proper dose levels are well knownin the art and within the skills of an ordinary physician.

Any known administration regimen for regulating the timing and sequenceof drug delivery may be used and repeated as necessary to effecttreatment in the inventive methods. The regimen may include pretreatmentand/or co-administration with additional therapeutic agent(s).

The inventive compounds can be administered alone or in combination withone or more additional therapeutic agent(s) for simultaneous, separate,or sequential use. Examples of an additional therapeutic agent include,without limitation, compounds of this invention; steroids (e.g.,hydrocortisones such as methylprednisolone); anti-inflammatory oranti-immune drug, such as methotrexate, azathioprine, cyclophosphamideor cyclosporin A; interferon-β; antibodies, such as anti-CD4 antibodies;agents which can reduce the risk of a second ischemic event, such asticlopidine; chemotherapeutic agents; immunotherapeutic compositions;electromagnetic radiosensitizers; and morphine. The inventive compoundsmay be co-administered with one or more additional therapeutic agent(s)either (i) together in a single formulation, or (ii) separately inindividual formulations designed for optimal release rates of theirrespective active agent.

Diagnostic Methods and Kit

This invention further provides a method for detecting in vitro or invivo a disease, disorder or condition where NAALADase levels arealtered, comprising:

(i) contacting a sample of bodily tissue or fluid with a compound offormula I, II or III, as defined above, wherein the compound binds toany NAALADase in the sample; and

(ii) measuring the amount of any NAALADase bound to the sample, whereinthe amount is diagnostic for the disease, disorder or condition.

Examples of a disease, disorder or condition that is detectable by theinventive method include, without limitation, neurological disorder,glutamate abnormality, neuropathy, pain, compulsive disorder, prostatedisease, cancer, TGF-β abnormality, Huntington's disease, diabetes,retinal disorder and glaucoma.

Examples of a bodily tissue or fluid that is used in the inventivemethod include, without limitation, prostate tissue, ejaculate, seminalvesicle fluid, prostatic fluid, urine, blood, saliva, tears, sweat,lymph and sputum.

The inventive compound may be labeled with a marker using techniquesknown in the art. Useful markers include, without limitation, enzymaticmarkers and imaging reagents. Examples of imaging reagents includeradiolabels, such as ¹³¹I, ¹¹¹In, ¹²³I, ⁹⁹Tc, ³²p, ¹²⁵I, ³H and ¹⁴C;fluorescent labels, such as fluorescein and rhodamine; andchemiluminescers, such as luciferin.

The amount of NAALADase can be measured using any technique known in theart. Examples of such technique include, without limitation, assays(such as immunometric, calorimetric, densitometric, spectrographic andchromatographic assays) and imaging techniques (such as magneticresonance spectroscopy (MRS), magnetic resonance imaging (MRI),single-photon emission computed tomography (SPECT) and positron emissiontomography (PET)).

This invention further provides a diagnostic kit for detecting adisease, disorder or condition where NAALADase levels are altered,comprising a compound of formula I, II or III, as defined above, labeledwith a marker. The kit may further comprise one or more bufferingagent(s), agent(s) for reducing background interference, controlreagent(s) and/or apparatus for detecting the disease, disorder orcondition.

This invention further provides a method for detecting a disease,disorder or condition where NAALADase levels are altered in an animal ora mammal, comprising:

(i) labeling a compound of formula I, II or III, as defined above, withan imaging reagent;

(ii) administering to the animal or mammal an effective amount of thelabeled compound;

(iii) allowing the labeled compound to localize and bind to NAALADasepresent in the animal or mammal; and

(iv) measuring the amount of NAALADase bound to the labeled compound,wherein the amount is diagnostic for the disease, disorder or condition.

The amount of NAALADase can be measured in vivo using any known imagingtechnique, as described above.

Incorporation by Reference

The relationship between NAALADase inhibitors and glutamate, and theeffectiveness of NAALADase inhibitors in treating and detecting variousdiseases, disorders and conditions have been discussed in U.S. Pat. Nos.5,672,592, 5,795,877, 5,804,602, 5,824,662, 5,863,536, 5,977,090,5,981,209, 6,011,021, 6,017,903, 6,025,344, 6,025,345, 6,046,180,6,228,888, 6,265,609, 6,372,726, 6,395,718, 6,444,657, 6,452,044,6,458,775 and 6,586,623; International Publications Nos. WO 01/91738, WO01/92274 and WO 03/057154; and other references generally known in theart. The present inventors hereby incorporate by reference, as thoughset forth herein in full, the entire contents of the aforementionedreferences, particularly their discussions, figures and data regardingthe effectiveness of NAALADase inhibitors in inhibiting angiogenesis; ineffecting TGF-β activity; in diagnosing a disease, disorder orcondition; and in treating glutamate abnormality, compulsive disorder,ischemia, spinal cord injury, demyelinating diseases, Parkinson'sdisease, ALS, alcohol dependence, nicotine dependence, cocainedependence, prostate disease, cancer, diabetic neuropathy, pain,schizophrenia, anxiety, anxiety disorder, memory impairment,Huntington's disease, diabetes, retinal disorders and glaucoma. Sincethe inventive compounds have been found by the present inventors toinhibit NAALADase activity or convert into compounds that inhibitNAALADase activity, they are expected to have the same uses as theNAALADase inhibitors disclosed in the patents and publicationsincorporated by reference.

Pharmaceutical Compositions

This invention further provides a pharmaceutical composition comprising:

(i) an effective amount of a compound of formula I, II or III; and

(ii) a pharmaceutically acceptable carrier.

The inventive pharmaceutical composition may comprise one or moreadditional pharmaceutically acceptable ingredient(s), including withoutlimitation one or more wetting agent(s), buffering agent(s), suspendingagent(s), lubricating agent(s), emulsifier(s), disintegrant(s),absorbent(s), preservative(s), surfactant(s), colorant(s), flavorant(s),sweetener(s) and additional therapeutic agent(s).

The inventive pharmaceutical composition may be formulated into solid orliquid form for the following: (1) oral administration as, for example,a drench (aqueous or non-aqueous solution or suspension), tablet (forexample, targeted for buccal, sublingual or systemic absorption), bolus,powder, granule, paste for application to the tongue, hard gelatincapsule, soft gelatin capsule, mouth spray, emulsion and microemulsion;(2) parenteral administration by, for example, subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution, suspension or sustained-release formulation; (3)topical application as, for example, a cream, ointment, orcontrolled-release patch or spray applied to the skin; (4) intravaginalor intrarectal administration as, for example, a pessary, cream or foam;(5) sublingual administration; (6) ocular administration; (7)transdermal administration; or (8) nasal administration.

EXAMPLES Example 1 Preparation of 3-(2-oxo-tetrahydro-thiopyran-3-yl)propionic acid

(Compound 1)

To a solution of 2-[3-(tritylthio)mercaptopropyl]pentanedioic acid (150g, 0.33 mol) in dichloromthane (500 mL) was added dropwisetrifluoroacetic acid (110 mL) over 30 minutes. After being stirred foran additional 30 minutes, a solution of triethylsilane (45 mL, 0.33 mol)in dichloromethane (50 mL) was added and the mixture was stirred at 45°C. for 1 hour. The volatiles were removed in vacuo and the residue wastriturated with hexanes (500 mL×2). The oily residue was dissolved intoluene (500 mL) containing 10-camphorsulfonic acid (14 g) and refluxedfor 6 hours. The liberated water was removed using a Dean-Storkazeotropic adapter. Toluene was then distilled off and the residue waspurified by silica gel chromatography (EtOAc/hexanes, 1/4). Subsequentrecrystallization from EtOAc/hexanes afforded 23.3 g of3-(2-oxotetrahydro-2H-thiopyran-3-yl)propanoic acid as a white solid(37% yield): ¹H NMR (CD₃OD) δ 1.65-1.78 (m, 2 H), 2.05-2.18 (m, 4 H),2.35-2.48 (m, 2 H), 2.65-2.74 (m, 1 H), 3.13-3.29 (m. 2 H); ¹³C NMR(CD₃OD) δ 23.3, 27.4, 29.3, 31.2, 32.3, 50.0, 177.0, 206.4. Analysiscalculated for C₈H₁₂O₃S: C, 51.04; H, 6.43; S, 17.03. Found: C, 51.08;H, 6.38; S, 17.16.

Example 2 Preparation of3-[2-oxotetrahydro-2H-thiopyran-3-yl)methyl]benzoic acid

A solution of 3-(2-carboxy-5-mercaptopentyl)benzoic acid (5.12 g, 20mmol) and 10-camphorsulfonic acid (500 mg) in toluene (30 mL) wasrefluxed for 6 hours. The liberated water was removed using a Dean-Storkazeotropic adapter. Toluene was then distilled off under reducedpressure and the residue was purified by silica gel chromatography(EtOAc/hexanes, 1:4). Fractions containing the product were collected,evaporated and recrystallized from EtOAc/hexanes to afford 3.2 g of3-[(2-oxotetrahydro-2H-thiopyran-3-yl)methyl]benzoic acid as a whitesolid (67% yield): ¹H NMR (CDCl₃) δ 1.58-1.68 (m, 1 H), 1.90-2.02 (m, 2H), 2.04-2.13 (m, 1 H), 2.68 (m, 1 H), 2.78-2.86 (m, 1 H), 3.08-3.19 (m,2 H), 3.37-3.44 (m, 1 H), 7.38-7.48 (m, 2 H), 7.93 (brs, 1 H), 7.94-8.02(m, 1 H); ¹³C NMR (CDCl₃) δ 22.6, 27.8, 31.1, 36.8, 52.0, 128.8, 129.2,129.8, 131.3, 135.3, 140.1, 172.6, 203.7. Analysis calculated forC₁₃H₁₄O₃S: C, 62.38; H, 5.64; S, 12.81. Found: C, 62.19; H, 5.65; S,12.59.

Example 3 Preparation of4-{[(1-oxo-3,4-dihydro-1H-isothiochromen-8-yl)oxy]methyl}benzoic acid

To a solution of 2-[(4-carboxybenzyl)oxyl-6-(2-mercaptoethyl)benzoicacid (1.66 g. 5.0 mmol) in ethanol (80 mL) were added 4% aq. NaOH (20mL) and a solution of benzylbromide (0.89 g, 5.2 mmol) in ethanol (20mL) at 0° C. The reaction mixture was stirred at 0° C. for 3 hours. Thesolvent was removed under reduced pressure and the residue was dissolvedin EtOAc. The organic solution was washed with 12 N HCl. The organiclayer was dried over MgSO₄ and concentrated to give 2.0 g of whitesolid. This solid was dissolved in trifluoroacetic anhydride (15 mL) andthe resulting mixture was stirred at 60° C. for 2 hours. The volatileswere removed in vacuo and the residue was dissolved in saturated aqueousNaHCO₃ at 0° C. followed by acidification with 12 N HCl. The resultingpercipitate was recovered by filtration and washed thoroughly withwater. The recovered solid was purified by silica gel chromatography(EtOAc/hexanes/AcOH, 1/1/0.02) to give 0.8 g of4-{[(1-oxo-3,4-dihydro-1H-isothiochromen-8-yl)oxy]methyl}benzoic acid asa white powder (54% yield): ¹H NMR (DMSO-d₆) δ 3.15-3.30 (m, 4 H), 5.26(s, 2 H), 7.02-7.07 (m, 1 H), 7.20-7.27 (m, 1 H), 7.53-7.60 (m, 1 H),7.66-7.72 (m, 2 H), 8.00-8.05 (m, 2 H); ¹³C NMR (DMSO-d₆) δ 28.7, 31.6,69.2, 113.2, 121.2, 121.5, 126.6, 123.3, 130,0, 134.0, 142,0, 143.8,156.2, 167.1, 187.2. Analysis calculated for C₁₃H₁₄O₃S: C, 62.38; H,5.64; S, 12.81. Found: C, 62.19; H, 5.65; S, 12.59.

Example 4 Preparation of3-(1-oxo-3,4-dihydro-1H-isothiochromen-8-yl)benzoic acid

By the method previously outlined in Example 3 but using3-(2-mercaptoethyl)-[1,1′-biphenyl]-2,3′-dicarboxylic acid was made3-(1-oxo-3,4-dihydro-1H-isothiochromen-8-yl)benzoic acid (34% yield): ¹HNMR (DMSO-d₆) δ 3.21-3.42 (m, 4 H), 7.27-7.37 (m, 1 H), 7.45-7.53 (m,3H), 7.57-7.64 (m, 1 H), 7.75-7.79 (m, 1 H), 7.87-7.94 (m, 1 H).

Example 5 Efficacy of NAALADase Inhibitors in Treating Retinal Disorders

Four groups of rats received daily insulin injections to maintain theirglucose levels at about 350 mg/dl. Starting at the onset ofhyperglycemia, NAALADase inhibitor 2-(3-sulfanylpropyl)-pentanedioicacid (Compound B) was administered daily for 6 months to one group ofBB/W rats at a dose of 10 mg/kg and to a second group of BB/W rats at adose of 30 mg/kg. A third group of BB/W rats and a fourth group ofnon-diabetic rats received daily vehicle treatment (50 mM Hepes bufferedsaline).

After 6 months of treatment with Compound B or vehicle treatment, therats were sacrificed and their eyes were removed. From each rat, one eyewas processed for elastase digest while the other eye was processed fortransmission electron microscopy (TEM) and basement membrane (BM)thickness.

Elastase Digests

Retinal digests were prepared using elastase on retinas as described inLayer, N., Invest Ophthalmol Vis Sci (1993) 34:2097. Eyes were removedfrom recently killed BB/W rats (n=30) and age-matched transgeniccontrols (n=12). The retinas (n=12) were fixed at room temperature byimmersing the whole eye (slit at limbus) in 4% (w/v) paraformaldehyde in50 mmol/L Na—K phosphate buffer with 8% sucrose. The fixed retinas wererinsed in deionized water and were incubated for 3 minutes in a 37° C.agitating water bath in 40 units/mL elastase in Na—K phosphate bufferwith 150 mmol/L NaCl and 5 mmol/L ethylenediamine tetraacetic acid(EDTA), pH 6.5. The tissues were washed overnight in 100 mmol/L Tris-HCL(pH 8.5) and then transferred to deionized water for removal of theloosened vitreous and digested neural elements by gentle agitation usingthe sides of closed forceps and the sides and ends of very fine brushes.After all loose tissues were removed, the retinas were incubated oncemore in fresh enzyme for 3 minutes and then subjected to a secondovernight wash at room temperature in Tns-HCl buffer. On the third day,the retinas were again transferred to deionized water for additionalremoval of digested neural elements. The vascular network that wascompletely free of nonvascular elements was mounted flat by flotation inCa²⁺ and Mg²⁺ free Dulbecco's Phosphate-Buffered Saline (PBS) onsiliconized slides (#S1308, Oncor, Gaithersburg, Md.). After air dryingin a dust free environment, the mounts of the retinal microvasculaturewere stained using periodic acid Schiff reaction and hematoxylincounterstaining, as described in Luna, L., ed. Manual of HistologicStaining Methods of the Armed Forces Institute of Pathology (1968)McGraw-Hill, New York, N.Y. The preparations were then examined by lightmicroscopy and photographed.

Endothelial/Pericyte (E/P) Ratios

The stained and intact retinal whole mounts were coded and counted, asdescribed in Cuthbertson, R., Invest Ophthalmol Vis Sci. (1986)27:1659-1664).

Ten fields at ×100 magnification were counted for endothelial andpericyte cells using previously described morphologic criteria (seeKuwabara, T., Arch Ophthalmol. (1960) 64:904-911). In every sample, atleast 200 cells were counted from the mid zone of the retina. Meanvalues for endothelial cell/pericyte (E/P) ratios were initiallycalculated in 3 retinas from each of the 4 groups of rats.

Evaluation of BM Thickness

Each eye was fixed in 4% glutaraldehyde and dissected free of sclera andchoroids, then trimmed and postfixed in 1% osmium tetroxide. Afterdehydration and embedding, thin sections were stained with uranylacetate and lead citrate. Initially, BM thickness of retinal capillariesfrom 3 non-diabetic rats receiving vehicle, 3 diabetic animals receiving10 mg/kg Compound B, and 3 diabetic rats receiving 30 mg/kg Compound Bwere compared with 3 diabetic rats receiving a vehicle. At least 10capillaries per eye from the inner nuclear and plexiform layers werephotographed at a magnification of 10,000×. Exact magnification wasdetermined for each set of negatives with a 28,800 line/inch calibrationgrid. Negatives were enlarged 3×. Measurements, to the nearest 0.25 mm,were made of the BM surrounding the endothelial cell and were takenperpendicular to the plane of the BM, as described in Bendayan, M., J.Electron Microsc Techn (1984) 1:243-270; and Gunderson, J. Microscopy(1980) 121:65-73). At least 20 measurements were taken for eachcapillary and the BM thickness was expressed as an average of 20measurements.

Statistical Analysis

Statistical analysis for comparison among groups was performed using oneway analysis of variance (ANOVA) and Student's t test. Significance wasdefined as a value of p<0.05. Values were reported as mean±standarderrors from the mean (SEM), unless otherwise noted.

Results of Elastase Digest Preparations and E/P Ratios

In intact whole mounts of retinal digests the endothelial cell nuclei,seen medially within the vessel wall, were large, oval, pale stainingand protruded lumenally. Pericyte nuclei, seen more laterally, were darkstaining, small, round and protruded prominently away from the vesselwall. E/P counts were taken from mid zones of the retinas. The attachedfigures show 27,000× magnified photographs of retinal blood vessels froma control, non-diabetic rat (FIG. 1), from a control, diabetic rat after6 months of treatment with a vehicle (FIG. 2), and from a diabetic ratafter 6 months of treatment with Compound B (FIG. 3).

NAALADase inhibition had no effect on blood glucose or body weight. Thehigh dose (30 mg/kg) treatment with Compound B resulted in a 29.0%reduction in BM thickness (diabetic vehicle=101.0±14.81 nm and diabeticNAALADase₃₀=71.7±4.07 nm), while the low dose (10 mg/kg) treatment withCompound B resulted in an 18.5% decrease in BM thickness(NAALADase₁₀=82.3±4.07 nm). The high dose treatment with Compound B alsoresulted in a 33.0% reduction of endothelial cell to pericyte ratios(diabetic vehicle=3.0±0.1 and NAALADase₃₀=2.0±0.9), while the low dosetreatment with Compound B resulted in a 16.7% reduction of the same cellratios (NAALADase₁₀=2.5±0.5). See TABLE II. TABLE II BM THICKNESS RATGROUP (nm) ± SD E/P RATIO NON-DIABETIC 56.3 ± 4.78  1.8 ± 0.07 CONTROLSDIABETIC VEHICLE   101 ± 14.81 3.0 ± 0.1 DIABETIC 30 MG/KG 71.7 ± 4.072.0 ± 0.4 NAAALADASE INHIBITOR DIABETIC 10 MG/KG 82.3 ± 4.07 2.5 ± 0.5NAALADASE INHIBITORConclusions

While the BB/W rats demonstrated an early change typically associatedwith diabetic retinopathy (pericyte loss and BM thickening), they didnot show significant numbers of microanuerysms also typical of diabeticretinopathy or areas of acellular capillaries usually seen in a moreadvanced disease. The retinopathy observed in BB/W rats has beenpreviously characterized in Chakrabarti, Diabetes (1989) 38:1181-1186.

The results show that treatment with a NAALADase inhibitor causesimprovement in retinal pathology of diabetic rats. Specifically, theNAALADase inhibitor prevented pericyte loss and BM thickening in retinalvessels.

Example 6 Protective Effect of NAALADase Inhibitors in Experimental Rat

Glaucoma

Experimental Protocol

All experiments complied with the Association for Research in Vision andOphthalmology Statement for the Use of Animals in Ophthalmic and VisionResearch. 82 male Brown Norway rats (Rattus norvegicus), each weighingapproximately 250 gm, were treated using procedures approved by theAnimal Care Committee of the Johns Hopkins University School ofMedicine. The rats were housed with a 12 hour light/12 hour dark cycleand fed ad libitum.

Experimental Glaucoma

Unilateral elevation of intraocular pressure (“IOP”) was produced in 56rats by microinjection of hypertonic saline into episcleral veins,following procedures described in Morrison, J. et al., IOVS (March 1998)39:526-531. Beginning on the day of IOP elevation, the rats were treateddaily with intraperitoneal injections of either a vehicle (23 rats with50 mM HEPES-buffered saline) or a NAALADase inhibitor (11 rats with 10mg/kg of Compound A and 22 rats with 10 mg/kg of Compound B). 11 salinetreated rats, 11 Compound A treated rats and 11 Compound B treated ratswere sacrificed at 8 weeks, and the remaining rats at 12 weeks, afterinitial IOP elevation.

Optic Nerve Transection

The optic nerve was transected unilaterally in 26 rats underintraperitoneal pentobarbital anesthesia. The conjunctiva was openedwith scissors and the optic nerve was exposed by traction on extraocularmuscles. The transection was performed with microscissors 5 mm posteriorto the globe, with specific attention to avoidance of injury to majorocular blood vessels. Immediately after transection, the retina wasexamined ophthalmoscopically to assure that the retinal arterial bloodsupply was not disrupted. The conjunctiva was closed with absorbablesuture and the eye dressed with antibiotic ointment. Beginning on theday of transection, the rats were treated daily with intraperitonealinjections of either a vehicle (9 rats with 50 mM HEPES-buffered saline)or a NAALADase inhibitor (8 rats with 10 mg/kg of Compound A and 9 ratswith 10 mg/kg of Compound B). 5 saline treated rats, 3 Compound Atreated rats and 4 Compound B treated rats were sacrificed at 2 weeks,and the remaining rats at 4 weeks, after transection.

Optic Nerve Counting

The rats were sacrificed by exsanguination under deep pentobarbitalanesthesia. They were perfused through the heart with 2%paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2,and the eyes with attached optic nerves were removed. Cross-sections ofthe optic nerves from both experimental (glaucoma or transection) andcontrol eyes was removed 1.5 mm posterior to the globe, 1 mm inthickness, and post-fixed in 2% osmium tetroxide in buffer. These wereprocessed into epoxy resin, sectioned at 1 micron and stained withtoluidine blue.

The area of each optic nerve cross-section was measured by outlining itsouter border at 10× magnification on an image analysis system (UniversalImaging Corp., Westchester, Pa.) with Synsys digital camera andMetamorph software. Three area measurements were taken and the meanvalue was determined. To measure the density and fiber diameterdistributions, images were captured with a 100× phase contrast objectivefrom 10 different areas of each nerve. These were edited to eliminatenon-neural objects and the size of each axon internal to the myelinsheath (its minimum diameter) and the density of axons/square mm werecalculated for each image and nerve. The mean density was multiplied bytotal nerve area to yield fiber number for each nerve. The total fibernumber in glaucoma or transection nerves was compared to the normal,fellow eye of each rat to yield a percent loss value. The number ofaxons counted among the 10 images was approximately 20% of the 80-90,000axons in normal rat nerves. The person measuring the number of axons wasmasked to the protocol conducted on the nerves.

Results

Experimental Glaucoma

The mean fiber percent difference in the saline-treated, control ratswas significantly lower in their glaucoma eyes compared to their normaleyes, with a mean fiber loss of 14.44±5.75% (n=11 rats; TABLE III) inthe 8 week follow-up group, and 8.15±7.84% in the 12 week follow-upgroup (n=12 rats; TABLE IV).

By contrast, there was no significant loss of fibers in either the 8week or 12 week NAALADase inhibitor-treated rats. The mean percent fiberloss in each NAALADase inhibitor-treated group was statistically lessthan the loss in the saline-treated, control groups (at 8 weeks, p=0.05for Compound A and p=0.02 for Compound B). TABLE III EXPERIMENTALGLAUCOMA RESULTS 8 Week IOP Integral Percent Group N Difference FIBERNUMBER Difference Compound A 11  85 ± 37.5 79156 ± 2436*  −1.82 ± 2.92Compound B 11 116 ± 33.2 80785 ± 2121** −0.82 ± 2.97 Control 11 104 ±26.4 68295 ± 4617  14.44 ± 5.75

TABLE IV EXPERIMENTAL GLAUCOMA RESULTS 12 Week IOP Integral PercentGroup N Difference FIBER NUMBER Difference Compound B 11 109 ± 45.290504 ± 1718 −3.21 ± 2.86 Control 12 158 ± 66.5 79827 ± 6783  8.15 ±7.84

-   IOP Integral Difference=difference in IOP exposure between glaucoma    eye and normal eye in each rat (mm Hg—days).-   Percent Difference=mean percent difference in fiber number between    glaucoma and normal eye in each rat (positive value indicates fewer    fibers in the glaucoma eye).-   The differences in IOP Integral Difference are not significant    (p>0.05). The differences in Percent Difference between drug-treated    and saline-treated, control rats at 8 weeks post insult are    significant (p=0.05* and p=0.02**).    Optic Nerve Transection

The experimental transection data suggest a slowing or rescue ofultimate retinal ganglion cell (RGC) death in rats treated withNAALADase inhibitors at 2 weeks after transection. At 2 weeks aftertransection, both drug-treated groups had more remaining RGC axons thandid the saline-treated, control group, judged either by absolute numberof fibers or percent difference between transected eye and normal eye ineach rat (TABLE V). Rats treated with Compound A and Compound B had,respectively, three times and twice as many remaining axons as thesaline-treated rats. All or nearly all RGC die within the first 2 monthsafter transection, regardless of any pharmacological treatment. Thus, by4 weeks after transection, more than 80% of RGC axons were gone in allgroups (TABLE VI). At 4 weeks after transection, there were nosignificant differences between the drug-treated rats and thesaline-treated rats. TABLE V OPTIC NERVE TRANSECTION 2 Weeks Survival NFiber Number Percent Difference Compound A 3 26,426 ± 23,025 65.3 ± 30.9Compound B 4 19,550 ± 11,383 75.3 ± 14.8 Control 5 8,220 ± 9,337 90.2 ±10.7

TABLE VI OPTIC NERVE TRANSECTION 4 Weeks Survival N Fiber Number PERCENTDIFFERENCE Compound A 5 13,599 ± 7,868 82.4 ± 8.9 Compound B 5  5,162 ±5,017 93.4 ± 6.2 Control 4 10,449 ± 8,157  86.9 ± 10.6

-   Percent Difference=mean percent difference in fiber number between    glaucoma and normal eye in each rat (positive value indicates fewer    fibers in the glaucoma eye).-   The differences in Percent Difference between drug-treated and    saline-treated, control rats are not statistically significant    (p=0.05).

Example 7 Effects of NAALADase Inhibitor on Development of MorphineTolerance

Experimental Protocol

Subjects

Male C57/BL mice (IMP, Lodz, Poland), 22-24 g of body weight weregroup-housed in the standard laboratory cages and kept in atemperature-controlled colony room (21±2° C.) with a 12-hour light/darkcycle (light on: 07:00, off: 19:00). Commercial food and tap water wereavailable ad libitum. Each experimental group consisted of 7-28 mice pertreatment. All mice were used only once.

Apparatus for Experiments 1-2

A standardized tail-flick analgesia meter (Columbus, Ohio, USA, model33), adjusted to sensitivity of “10” with radiant heat source andconnected to an automatic timer was used to assess antinociceptiveresponses. The intensity of the heat stimulus was adjusted so that thebaseline tail-flick latency was ˜3 seconds. A maximum latency of 10seconds (i.e., cut-off) was used to minimize damage to the tail. Thetail withdrawal latency was measured from the start of heat stimulusuntil the mouse exhibited a flick of the tail. Each response assessmentconsisted of two separate measurements taken at different portions ofthe tail (spaced by 1.5-2 cm) and separated by 15 seconds. The mean ofthese responses was used for subsequent comparisons.

Morphine antinociceptive potency was investigated with the use ofcumulative dose-response curves that allowed for minimization of theanimal number used (Paronis and Holtzman 1991). After adaptation andbaseline trials, each mouse was injected s.c. with a low dose ofmorphine (1 mg/kg). Thirty min later, the mouse was retested andinjected with the next dose of morphine that was increased by quarter ofa log unit. Thus, because the initial dose of morphine was 1.0 mg/kg,the next dose was 1.78 mg/kg, for a cumulative dose of 2.8 mg/kg. Thisprocedure continued until either the mouse did not move his tail withinthe cut-off time or until there was a plateauing of the dose-responsecurve, so that the latency did not increase from one dose to the next.Each analgesic responder was not subjected to further tail flickassessments but was injected with the subsequent dose of morphine sothat every animal received the same total dose of morphine during agiven test.

Effects on Morphine Tolerance (Experiment 1) and Acute Effects in theTail-Flick Test (Experiment 2)

Experiment 1 was carried out to investigate the effect of 2-PMPA on thedevelopment of morphine tolerance. On day 1 (test #1), the firstmeasurement of morphine antinociceptive potency was performed, followedby 6 days of bid morphine injections (10 mg/kg, s.c., 7:30 and 17:30)(Elliott et al. 1994; Popik et al. 2000b). Pretreatment with2-(phosphonomethyl)pentanedioic acid (2-PMPA; 30, 50 or 100 mg/kg, i.p.)or memantine (7.5 mg/kg, s.c., a “positive control”) was given at 30minutes prior to each morphine dose on days 2-7. On day 8 (test #2), thesecond measurement of morphine antinociceptive potency was carried out.The degree of morphine tolerance was assessed by comparing the morphineantinociceptive potencies (cumulative dose-response curves) obtained intests #1 and #2.

Experiment 2 was designed to determine whether 2-PMPA might itselfproduce antinociceptive effects and/or affect the antinociceptiveeffects of morphine. Morphine (1.5 or 3 mg/kg, s.c.) was administered 30min after injection of 100 mg/kg of 2-PMPA or placebo, administered i.p.The 3 mg/kg dose of morphine corresponds to the antinociceptive ED₅₀dose in these test conditions (data not shown).

Results

Effects of NAALADase Inhibitor on Development of Morphine Tolerance

(Experiment 1)

There were no differences in antinociceptive morphine ED₅₀ values ontest #1 among groups (TABLE VII). Treatment with 10 mg/kg bid ofmorphine produced 6.44 fold increase in the ED₅₀ values as determined ontest #2. In contrast, pretreatment with memantine, 50 or 100 (but not30) mg/kg of 2-PMPA given prior to each dose of morphine attenuated thedevelopment of morphine tolerance. The effects of 2-PMPA were related tothe dose. This was evidenced by a significant decrease in both test #2ED₅₀ values (statistically significant for the dose 100 mg/kg) andantinociceptive morphine fold shifts of 2-PMPA for the doses of 100 and50 mg/kg, as compared with the control group that receivedplacebo+morphine (Table VII). Similarly, memantine (7.5 mg/kg) producedan inhibition of morphine tolerance. TABLE VII EFFECTS OF 2-PMPA ANDMEMANTINE ON THE DEVELOPMENT OF TOLERANCE TO MORPHINE Treatment/ Test #1Test #2 dose mg/kg (N) ED₅₀ ED₅₀ Fold Shift Placebo + 1.49 ± 0.26 8.85 ±2.22  6.44 ± 1.17  Morphine (8) Placebo + 2.23 ± 0.42 3.28 ± 0.47* 1.70± 0.29* Placebo (8) 2-PMPA 30 + 2.00 ± 0.43 9.47 ± 2.13  5.20 ± 1.26 Morphine (9) 2-PMPA 50 + 1.87 ± 0.34 5.41 ± 1.11  3.20 ± 0.66* Morphine(9) 2-PMPA 100 + 1.59 ± 0.30 3.49 ± 0.83* 2.70 ± 0.57* Morphine (10)Memantine 7.5 + 1.51 ± 0.29 3.52 ± 0.88* 2.60 ± 0.49* Morphine (8)ANOVA: F(5, 46) = 0.71; ns 3.891; P < 0.01 4.555; P < 0.01

Presented are mean ED₅₀ values with±SEM determined during test #1(pre-morphine) and test #2 (post-morphine) as well as resulting foldshifts. Asterisks (*) indicate a statistically significant differencecompared to the Placebo+Morphine group that received saline and morphineduring the development of morphine tolerance (*p<0.05, Newman Keul'stest).

Effects of 2-PMPA on the Tail-Flick Response and Antinociceptive Effectsof Morphine

(Experiment 2)

Analysis of areas under curve (AUC) revealed that treatment withplacebo+1.5 and 3 mg/kg of morphine produced statistically significantlylonger tail-flick responses compared to placebo+placebo treatment. Incontrast, 100 mg/kg of 2-PMPA+placebo treatment did not affecttail-flick responses as compared to placebo+placebo treatment. Moreover,this dose of 2-PMPA did not affect antinociceptive effects of 1.5 or 3mg/kg of morphine (FIG. 4).

Presented in FIG. 4 are the time courses of tail-flick responses of micetreated with combination of 2-PMPA and morphine. The N is given inbrackets. Presented in FIG. 5 are mean±S.E.M. Area Under Curve (AUC)values calculated on the same data. One way ANOVA F(5,48)=19.28,P<0.0001 and post-hoc Newman-Keul's test revealed that the treatmentwith placebo+morphine 1.5 mg/kg and with 100 mg/kg 2-PMPA+morphine 1.5mg/kg differed significantly (**, P<0.01) from placebo+placebotreatment. Similarly, treatment with placebo+morphine 3 mg/kg and thatwith 100 mg/kg 2-PMPA+morphine 3 mg/kg differed significantly (***,P<0.001) from placebo+placebo treatment. Effects of 100 mg/kg of2-PMPA+placebo treatment did not differ from placebo+placebo treatment.Effects of placebo+respective doses of morphine did not differ from theeffects of 2-PMPA+respective doses of morphine.

Example 8

A patient is suffering from any disease, disorder or condition whereNAALADase levels are altered, including any of the diseases, disordersor conditions described above. The patient may then be administered aneffective amount of an inventive compound. It is expected that aftersuch treatment, the patient would not suffer any significant injury dueto, would be protected from further injury due to, or would recover fromthe disease, disorder or condition.

All publications, patents and patent applications identified above areherein incorporated by reference.

The invention being thus described, it will be apparent to those skilledin the art that the same may be varied in many ways without departingfrom the spirit and scope of the invention. Such variations are includedwithin the scope of the invention to be claimed.

1. A compound of formula I

or a pharmaceutically acceptable salt, an optical isomer or a mixture ofoptical isomers of the compound, wherein: X is C₁-C₄ alkylene or Ar; Aris phenylene which is unsubstituted or substituted with one or moresubstituent(s) independently selected from carboxy, halo, nitro, C₁-C₄alkyl, C₁-C₄ alkoxy, phenyl, phenoxy and benzyloxy; L is a bond,—CR¹R²—, —O—, —S—, or —NH; Y is —CR³R⁴—; Z is —(CR⁵R⁶)₂—; and R¹, R²,R³, R⁴, R⁵ and R⁶ are independently hydrogen, C₁-C₄ alkyl or C₂-C₄alkenyl, wherein the alkyl or alkenyl is unsubstituted or substitutedwith one or more substituent(s).
 2. The compound of claim 1, wherein Lis —CR¹R²—.
 3. The compound of claim 1, wherein R¹, R², R³, R⁴, R⁵ andR⁶ are each hydrogen.
 4. The compound of claim 1, wherein X is Ar. 5.The compound of claim 1, wherein: L is —CR¹R²—; R¹, R², R³, R⁴, R⁵ andR⁶ are each hydrogen; and X is Ar.
 6. The compound of claim 5, whereinAr is phenylene substituted with one or more substituent(s)independently selected from carboxy, halo, nitro, C₁-C₄ alkyl, C₁-C₄alkoxy, phenyl, phenoxy and benzyloxy.
 7. The compound of claim 5,wherein Ar is phenylene substituted with carboxy.
 8. The compound ofclaim 5, wherein Ar is phenylene substituted with halo.
 9. The compoundof claim 5, wherein Ar is phenylene substituted with nitro.
 10. Thecompound of claim 5, wherein Ar is phenylene substituted with C₁-C₄alkyl.
 11. The compound of claim 5, wherein Ar is phenylene substitutedwith C₁-C₄ alkoxy.
 12. A method for treating peripheral neuropathy,comprising administering to a mammal in need thereof an effective amountof a compound of claim 1, or a pharmaceutically acceptable salt, anoptical isomer or a mixture of optical isomers of the compound.
 13. Themethod of claim 12, wherein the peripheral neuropathy is HIV-, chemical-or vitamin-induced.
 14. A pharmaceutical composition comprising: (i) aneffective amount of a compound of claim 1, or a pharmaceuticallyacceptable salt, an optical isomer or a mixture of optical isomers ofthe compound; and (ii) a pharmaceutically acceptable carrier.